Reconstitution of Cu2+-depleted bovine serum amine oxidase with Co2+.
Two different Cu2+-depleted derivatives of bovine serum amine oxidase (BSAO) have recently been prepared, which contain about 0.5 mol/dimer of phenylhydrazine-reactive topa quinone (TPQ) cofactor and, depending on the reagents used, about 0.2 or 0.7 residual Cu2+/dimer [Agostinelli, De Matteis, Sinibaldi, Mondovi and Morpurgo (1997) Biochem. J. 324, 497-501]. The benzylamine oxidase activity of both derivatives was <5% and increased up to approximately 20% on incorporation of Co2+, irrespective of the residual Cu2+ content, which was unaffected by the treatment according to atomic absorption and ESR spectroscopy. The residual Cu2+ ions appeared to be distributed one per dimer and to be bound to inactive subunits, whereas Co2+ was bound to active subunits. The change in the active site had an appreciable influence on the kinetic behaviour. With several amines, the kinetic parameters, Km and kc, measured for Co2+-BSAO were different from those for native BSAO. This excludes the possibility that the catalytic activity was due to residual Cu2+. Furthermore, Co2+ restored to nearly native level the intensity of the TPQ 480 nm band and the reactions with phenylhydrazine or benzylhydrazine, which had been slowed down or abolished, respectively, in Cu2+-depleted samples. The CD spectrum, measured for the derivative with low Cu2+ content, was compatible with Co2+ binding to the copper site. The amine oxidase activity of the Co2+ derivative, which cannot form a semiquinone radical as an intermediate of the catalytic reaction, strongly suggests that the Cu+-semiquinone is not an obligatory intermediate of BSAO catalytic pathway.
Agostinelli
E
E
Dipartimento di Scienze Biochimiche 'A. Rossi Fanelli' and Centro di Biologia Molecolare del Consiglio Nazionale delle Ricerche, Università di Roma 'La Sapienza', P.le A. Moro, 5, 00185 Roma, Italy.
De Matteis
G
G
Mondovì
B
B
Morpurgo
L
L
D006631:Amine Oxidase (Copper-Containing); D000818:Animals; D001596:Benzylamines / Q000737:chemistry; D002384:Catalysis; D002417:Cattle; D003035:Cobalt / Q000737:chemistry; D003300:Copper / Q000737:chemistry; D003486:Cyanides / Q000737:chemistry; D004227:Dithionite / Q000737:chemistry; D008667:Metalloproteins / Q000737:chemistry; D000587:Oxidoreductases Acting on CH-NH Group Donors / Q000737:chemistry*; D013329:Structure-Activity Relationship
D001596:Benzylamines; D003486:Cyanides; D008667:Metalloproteins; D004227:Dithionite; D003035:Cobalt; D003300:Copper; D006631:Amine Oxidase (Copper-Containing); D000587:Oxidoreductases Acting on CH-NH Group Donors